Lenalidomide and thalidomide immunoassays

ABSTRACT

Novel conjugates and immunogens derived from lenalidomide and antibodies generated by these immunogens are useful in immunoassays for the quantification and monitoring of thalidomide and lenalidomide in biological fluids.

FIELD OF THE INVENTION

This invention relates to the field of immunoassays for determining thepresence and/or quantifying the amount of lenalidomide and thalidomidein human biological fluids in order to rapidly determine optimal drugconcentrations during treatment.

BACKGROUND OF THE INVENTION

Cancer is a term used to describe a group of malignancies that all sharethe common trait of developing when cells in a part of the body begin togrow out of control. Most cancers form as tumors, but can also manifestin the blood and circulate through other tissues where they grow. Cancermalignancies are most commonly treated with a combination of surgery,chemotherapy, and/or radiation therapy. The type of treatment used totreat a specific cancer depends upon several factors including the typeof cancer malignancy and the stage during which it was diagnosed.

The chemotherapeutic agent whose common chemical name is thalidomide hasthe following formula:

The chemotherapeutic agent whose common chemical name is lenalidomidehas the following formula:

Thalidomide possesses immunomodulatory, anti-inflammatory andanti-angiogenic properties. The immunomodulatory and anti-inflammatoryproperties may be related to suppression of excessive tumor necrosisfactor-alpha production through degradation of mRNA encoding the factor(Moreira, J Exp Med, 177(6): 1675-80, 1993). Other immunomodulatory andanti-inflammatory properties of thalidomide may include suppression ofmacrophage involvement in prostaglandin synthesis, and modulation ofinterleukin-10 and interleukin-12 production by peripheral bloodmononuclear cells. The combination of anti-inflammatory andanti-angiogenic properties makes thalidomide a novel therapeutic agentwith significant potential in treating a wide variety of diseases (Teo,Clin Pharmacokinet, 43(5): 311-27, 2004). A number of recent clinicaltrials have demonstrated therapeutic effect of thalidomide in patientswith multiple myeloma, renal carcinoma and glioblastoma multiforme(Singhal, N Engl J Med, 341(21): 1565-71, 1999; Marx, J Neurooncol,54(1): 31-8, 2001). Currently, thalidomide is approved for treatment ofpatients with newly diagnosed multiple myeloma and for acute treatmentof erythema nodosum leprosum (Package-insert-Thalidomide, Celgene Corp.,2009).

Lenalidomide is a thalidomide derivative with immunomodulatory,anti-proliferative, and anti-angiogenic properties. Lenalidomide exertsdirect anti-proliferative effect on multiple myeloma cells by inducingcell cycle arrest and apoptosis (Armoiry, J Clin Pharm Ther, 33(3):219-26, 2008). Lenalidomide is approved for treatment of patients withmultiple myeloma and myelodysplastic syndromes associated with adeletion 5q cytogenetic abnormality (Package-insert-Revlimid, CelgeneCorp., 2009).

The mechanisms of action and metabolic pathways of thalidomide andlenalidomide are not fully characterized yet. In vivo, both drugs canundergo non-enzymatic hydrolysis and enzymatic metabolism producing amultitude of metabolites, but none of those compounds were found to beresponsible for thalidomide therapeutic effect Lepper, Curr Drug Metab,7(6): 677-85, 2006).

Thalidomide and lenalidomide exhibit significant variability in plasmaconcentrations. A phase I study of pharmacokinetic effects ofthalidomide in HIV patients has demonstrated a wide range of maximumdrug concentration C_(max) (2.8±2.6 mg/L) and half-life time t_(1/2)(5.9±2.3 hours) (Wohl, J Infect Dis, 185(9): 1359-63, 2002).Administration of thalidomide to healthy subjects resulted in up to 52%variability in C_(max) and up to 37% variability in to(Package-insert-Thalidomide, Celgene Corp., 2009). A Phase I trial oflenalidomide in patients with central nervous system tumors has revealedup to 78% variability in C_(max) and up to 122% variability in to (Fine,Clin Cancer Res, 13(23): 7101-6, 2007).

Since efficacy of thalidomide and lenalidomide is improved at higherconcentration levels and the drugs exhibit wide intra- and inter-patientpharmacokinetic variability monitoring concentrations of these drugs inblood and adjusting to target levels would be of value in increasingefficacy and minimizing toxicity. The degree of intra- andinter-individual pharmacokinetic variability of thalidomide andlenalidomide is impacted by many factors, including:

-   -   Age    -   Weight    -   Organ function    -   Drug-drug interaction    -   Genetic regulation    -   Compliance

As a result of this variability, equal doses of the same drug indifferent individuals can result in dramatically different clinicaloutcomes. The effectiveness of the same dosage of thalidomide andlenalidomide varies significantly based upon individual drug clearanceand the ultimate serum drug concentration in the patient. Therapeuticdrug management would provide the clinician with insight on patientvariation in drug administration. With therapeutic drug management, drugdosages could be individualized to the patient, and the chances ofeffectively treating the disorder without the unwanted side effectswould be much higher.

Routine therapeutic drug management of thalidomide and lenalidomidewould require the availability of simple automated tests adaptable togeneral laboratory equipment. The use of liquid chromatography (LC) withUV or mass spectroscopy detection to determine the concentration ofthalidomide and lenalidomide in human blood and plasma has beendescribed (Tohnya, J Chromatogr B Analyt Technol Biomed Life Sci,811(2): 135-41, 2004; Chen, J Clin Pharmacol, 47(12): 1466-75, 2007;Teo, J Clin Pharmacol, 39(11): 1162-8, 1999). These methods are laborintensive, requiring liquid-liquid or solid phase extractions, useexpensive equipment and are not amenable to routine clinical laboratoryuse. To date, there are no immunoassays for measuring lenalidomideand/or thalidomide in human biological fluids of patients treated withthese chemotherapeutic agents.

As seen from the foregoing, there are no immunoassays for determiningthe presence and/or quantifying the amount of thalidomide andlenalidomide in human biological fluids. Routine therapeutic drugmanagement of thalidomide and lenalidomide by immunoassays would providesimple automated tests adapted to standard laboratory equipment.However, in order to provide such immunoassays, antibodies specific tothalidomide and lenalidomide must be produced. The derivatives andimmunogen used in this assay must impart through these correspondingantibodies produced specific reactivity to thalidomide and lenalidomide.

SUMMARY OF INVENTION

In accordance with this invention, a new class of antibodies have beenproduced which are substantially reactive to thalidomide andlenalidomide and can be used in the same immunoassay to determine thepresence and/or quantify the amount of thalidomide and lenalidomide inpatients' samples treated with these chemotherapeutic drugs.

It has been found that by using immunogens which are conjugates of animmunogenic carrier containing polyamine polymer with a compound of theformula:

-   -   wherein B is —C(═O)—CH₂—, —C(═O)—NH—CH₂—, —C(═O)—O—CH₂— or —CH₂—        -   Y is an organic spacing group;        -   p is an integer from 0 to 1;        -   X is a terminal functional group capable of binding to said            polyamine polymer,            antibodies are produced which are specific for lenalidomide            as well as mixtures of lenalidomide with thalidomide and are            non reactive or non binding with pharmaceutically inactive            metabolites of both thalidomide and lenalidomide.

The provision of these antibodies which are selectively reactive witheither lenalidomide or a mixture of thalidomide and lenalidomide, allowsone to produce an immunoassay which can specifically detect and quantifyso as to monitor thalidomide and lenalidomide in the fluid samples ofpatients being treated with either thalidomide or lenalidomide. Alsoincluded within this invention are reagents and kits for saidimmunoassay.

DETAILED DESCRIPTION

In accordance with this invention, a new class of antibodies is providedwhich selectively binds to lenalidomide or mixtures of lenalidomide withthalidomide and is not cross reactive with pharmaceutically inactivemetabolites of either thalidomide and lenalidomide. It has beendiscovered that through the use of these derivatives of lenalidomide offormula III as immunogens, this new class of antibodies of thisinvention is provided. It is through the use of these antibodies that animmunoassay, including reagents and kits for such immunoassay fordetecting and/or quantifying thalidomide and lenalidomide in blood,plasma or other body fluid samples has been developed.

In accordance with this invention a new class of reagents is providedwhich can be used in either of these immunoassays for detecting and/orquantifying thalidomide or lenalidomide in samples. This reagent is aconjugate of a carrier with a ligand having the formula:

-   -   wherein B, Y and P are as above and X² is a terminal functional        group capable of binding to said carrier.

By use of this immunoassay, the presence and amount of thalidomide orlenalidomide in body fluid samples of patients being treated with eithertherapeutic agent can be detected and/or quantified. In this manner, apatient being treated with thalidomide or lenalidomide can be monitoredduring therapy and the treatment adjusted in accordance with saidmonitoring by using antibodies produced by the immunogen of formula IIIand the conjugate of Formula III-A. By means of this invention oneachieves the therapeutic drug management of thalidomide and lenalidomidein patients being treated with either thalidomide or lenalidomide astherapeutic agents. The therapeutic agents to be detected and/orquantified are thalidomide of formula I and lenalidomide of formula II.

The provision of the conjugates of formulae III-A as a reagent in theimmunoassay and the immunogen of Formula III conjugated with animmunogenic carrier provides antibodies and reagents which can beutilized in immunoassays to detect and/or quantify the chemotherapeuticagents lenalidomide and thalidomide. These reagents and the antibodiesproduced in accordance with this invention can be utilized both inimmunoassays for detecting and quantifying lenalidomide or thalidomide.In general, patients are treated with one and not both of thesechemotherapeutic agents. Therefore, an antibody or reagent which isselectively reactive against both lenalidomide and thalidomide can beutilized in these immunoassays to detect either lenalidomide orthalidomide. This is true, since a patient treated lenalidomide is notgenerally treated with thalidomide and a patient treated withthalidomide is not generally treated lenalidomide. Therefore, thereagents and antibodies of this invention can be used in either of thesetwo immunoassays to separately detect and/or quantify these twochemotherapeutic agents.

As set forth hereinbefore, the antibodies that can be produced by theimmunogen of formula III are selectively reactive with lenalidomide andmixtures of thalidomide with lenalidomide and can be used in either animmunoassay for thalidomide or for lenalidomide. While these antibodiesare selectively reactive with both thalidomide and lenalidomide, inorder to be used in an immunoassay for thalidomide, the antibody shouldhave a selective reactivity of at least about 10%, preferably at leastabout 40%, for thalidomide, based upon its combined reactivity with boththalidomide and lenalidomide. In accordance with this invention,antibodies can be produced utilizing the immunogen of formula III havingreactivity with thalidomide of at least about 10%, and at most about50%, based upon their reactivity with both lenalidomide and thalidomide.

On the other hand for utilizing an antibody in an immunoassay forlenalidomide, any of the antibodies produced by the immunogen of formulaIII having a selective reactivity with lenalidomide or with bothlenalidomide and thalidomide can be used. In accordance with thisinvention antibodies which are selectively reactive with lenalidomidebased upon their reactivity with thalidomide and lenalidomide orselectivity reactive with both lenalidomide and thalidomide can beproduced by means of the immunogen of formula III. In accordance withthis invention an antibody which is selectively reactive withlenalidomide and not thalidomide, i.e., antibodies having 100% selectivereactivity with lenalidomide based upon their selective reactivity withboth lenalidomide and thalidomide can be produced by use of theimmunogen of formula III. The antibodies having substantially 100%selective reactivity with lenalidomide and substantially no selectivereactivity with thalidomide are especially preferred for use in theimmunoassay for lenalidomide.

The reagents utilized in the assays of this invention are conjugates ofa polymeric carrier with the compounds of formula III-A. Theseconjugates are competitive binding partners with the thalidomide orlenalidomide present in the sample for the binding with the antibodiesof this invention. Therefore, the amount of this conjugate reagent whichbinds to the antibody will be inversely proportional to the amount ofthalidomide or lenalidomide in the sample. In accordance with thisinvention, the assay utilizes any conventional measuring means fordetecting and measuring the amount of said conjugate which is bound orunbound to the antibody. Through the use of said means, the amount ofthe bound or unbound conjugate can be determined. Generally, the amountof thalidomide and lenalidomide in a sample is determined by correlatingthe measured amount of the bound or unbound conjugate produced by thethalidomide or lenalidomide in the sample with values of the bound orunbound conjugate determined from a standard or calibration curveobtained with samples containing known amounts of thalidomide orlenalidomide, which known amounts are in the range expected for thesample to be tested. These studies for producing calibration curves aredetermined using the same immunoassay procedure as used for the sample.

The conjugates, as well as the immunogens, are prepared from compoundsof the formula III. When in the conjugates or immunogens, the carrierand the polyamine polymer are linked to ligand portions of the compoundsof formula III, this ligand portions has the formula:

wherein X′ is —CH₂— or a functional linking group;and Y, p and B, are as above

This ligand portion may be linked to one or more active sites on thecarrier of the conjugate or the immunogen. Generally these carrierscontain polymers, most preferably polyamine polymers having a reactiveamino group. In forming the conjugates especially the immunogen, X′ ispreferably a functional group which can react with an amino group.However with respect to the reagent used in the immunoassay, X′ can beany functional group which can react with any conventional carrier. Whenthe compound of formula III is used to make immunogens, X′ in thecompound of formula III is preferably any functional group capable ofbinding or linking to a polyamine polymer.

DEFINITIONS

Throughout this description the following definitions are to beunderstood:

The term “alkylene” designates a divalent saturated straight or branchchain hydrocarbon substituent containing from one to ten carbon atoms

The terms “immunogen” and “immunogenic” refer to substances capable ofeliciting, producing, or generating an immune response in an organism.

The term “conjugate” refers to any substance formed from the joiningtogether of two parts. Representative conjugates in accordance with thepresent invention include those formed by the joining together of asmall molecule, such as the compound of formula III or the compound offormula III-A and a large molecule, such as a carrier, preferablycarriers which comprise a polyamine polymer, particularly a protein. Inthe conjugate the small molecule may be joined or linked at one or moreactive sites on the large molecule. The term conjugate includes the termimmunogen. In the conjugates used as reagents the carrier can be anycarrier and X can be any functional group which can be linked to acarrier. In the immunogen the carrier is a polyamine polymer and X isany functional group capable of linking to a polyamine polymer.

“Haptens” are partial or incomplete antigens. They are protein-freesubstances, mostly low molecular weight substances, which are notcapable of stimulating antibody formation, but which do react withantibodies. The latter are formed by coupling a hapten to a highmolecular weight immunogenic carrier and then injecting this coupledproduct, i.e., immunogen, into a human or animal subject. The hapten ofthis invention is lenalidomide (II).

As used herein, a “spacing group” or “spacer” refers to a portion of achemical structure which connects two or more substructures such ashaptens, carriers, immunogens, labels, or tracer through a CH₂ orfunctional linking group. These spacer groups will be enumeratedhereinafter in this application. The atoms of a spacing group and theatoms of a chain within the spacing group are themselves connected bychemical bonds. Among the preferred spacers are straight or branched,saturated or unsaturated, carbon chains. Theses carbon chains may alsoinclude one or more heteroatoms within the chain or at termini of thechains. By “heteroatoms” is meant atoms other than carbon which arechosen from the group consisting of oxygen, nitrogen and sulfur. Spacinggroups may also include cyclic or aromatic groups as part of the chainor as a substitution on one of the atoms in the chain.

The number of atoms in the spacing group is determined by counting theatoms other than hydrogen. The number of atoms in a chain within aspacing group is determined by counting the number of atoms other thanhydrogen along the shortest route between the substructures beingconnected. A functional linking group may be used to activate, e.g.,provide an available functional site on, a hapten or spacing group forsynthesizing a conjugate of a hapten with a label or carrier orpolyamine polymer.

An “immunogenic carrier,” as the terms are used herein, is animmunogenic substance, commonly a protein, that can join with a hapten,in this case lenalidomide or the lenalidomide derivatives hereinbeforedescribed, thereby enabling these hapten derivatives to induce an immuneresponse and elicit the production of antibodies that can bindspecifically with these haptens and to thalidomide. The immunogeniccarriers and the linking groups will be enumerated hereinafter in thisapplication. Among the immunogenic carrier substances are includedproteins, glycoproteins, complex polyamino-polysaccharides, particles,and nucleic acids that are recognized as foreign and thereby elicit animmunologic response from the host. The polyamino-polysaccharides may beprepared from polysaccharides using any of the conventional means knownfor this preparation.

Also various protein types may be employed as a poly (amino acid)immunogenic carrier. These types include albumins, serum proteins,lipoproteins, etc. Illustrative proteins include bovine serum albumin(BSA), keyhole limpet hemocyanin (KLH), egg ovalbumin, bovinethyroglobulin (BTG) etc. Alternatively, synthetic poly(amino acids) maybe utilized.

Immunogenic carriers can also include polyamino-polysaccharides, whichare high molecular weight polymers built up by repeated condensations ofmonosaccharides. Examples of polysaccharides are starches, glycogen,cellulose, carbohydrate gums such as gum arabic, agar, and so forth. Thepolysaccharide also contains polyamino acid residues and/or lipidresidues.

The immunogenic carrier can also be a poly (nucleic acid) either aloneor conjugated to one of the above mentioned poly(amino acids) orpolysaccharides.

The immunogenic carrier can also include solid particles. The particlesare generally at least about 0.02 microns (μm) and not more than about100 μm, and usually about 0.05 μm to 10 μm in diameter. The particle canbe organic or inorganic, swellable or non-swellable, porous ornon-porous, optimally of a density approximating water, generally fromabout 0.7 to 1.5 g/mL, and composed of material that can be transparent,partially transparent, or opaque. The particles can be biologicalmaterials such as cells and microorganisms, including non-limitingexamples such as erythrocytes, leukocytes, lymphocytes, hybridomas,Streptococcus, Staphylococcus aureus, E. coli, and viruses. Theparticles can also be comprised of organic and inorganic polymers,liposomes, latex, phospholipid vesicles, or lipoproteins.

“Poly(amino acid)” or “polypeptide” is a polyamide formed from aminoacids. Poly(amino acids) will generally range from about 2,000 molecularweight, having no upper molecular weight limit, normally being less than10,000,000 and usually not more than about 600,000 daltons. There willusually be different ranges, depending on whether an immunogenic carrieror an enzyme is involved.

A “peptide” is any compound formed by the linkage of two or more aminoacids by amide (peptide) bonds, usually a polymer of α-amino acids inwhich the α-carboxyl group of each amino acid residue is linked to theα-amino group of the next residue in a linear chain. The terms peptide,polypeptide and poly(amino acid) are used synonymously herein to referto this class of compounds without restriction as to size. The largestmembers of this class are referred to as proteins.

A “label,” “detector molecule,” or “tracer” is any molecule whichproduces, or can be induced to produce, a detectable signal. The labelcan be conjugated to an analyte, immunogen, antibody, or to anothermolecule such as a receptor or a molecule that can bind to a receptorsuch as a ligand, particularly a hapten. Non-limiting examples of labelsinclude radioactive isotopes, enzymes, enzyme fragments, enzymesubstrates, enzyme inhibitors, coenzymes, catalysts, fluorophores, dyes,chemiluminescers, luminescers, or sensitizers; a non-magnetic ormagnetic particle, a solid support, a liposome, a ligand, or a receptor.

The term “antibody” refers to a specific protein binding partner for anantigen and is any substance, or group of substances, which has aspecific binding affinity for an antigen to the exclusion of othersubstances. The generic term antibody subsumes polyclonal antibodies,monoclonal antibodies and antibody fragments.

The term “derivative” refers to a chemical compound or molecule madefrom a parent compound by one or more chemical reactions.

The term “carrier” refers to solid particles and/or polymeric polymerssuch as immunogenic polymers such as those mentioned above. Where thecarrier is a solid particle, the solid particle may be bound, coatedwith or otherwise attached to the polymeric material which preferably isa polyamine polymer to provide one or more reactive sites for bonding tothe terminal functional group X in the compounds of the formula III.

The term “reagent kit,” or “test kit,” refers to an assembly ofmaterials that are used in performing an assay. The reagents can beprovided in packaged combination in the same or in separate containers,depending on their cross-reactivities and stabilities, and in liquid orin lyophilized form. The amounts and proportions of reagents provided inthe kit can be selected so as to provide optimum results for aparticular application. A reagent kit embodying features of the presentinvention comprises antibodies specific for the compounds of formula Iand formula II. The kit may further comprise ligands of the analyte andcalibration and control materials. The reagents may remain in liquidform or may be lyophilized.

The phrase “calibration and control materials” refers to any standard orreference material containing a known amount of a drug to be measured.The concentration of drug is calculated by comparing the resultsobtained for the unknown specimen with the results obtained for thestandard. This is commonly done by constructing a calibration curve.

The term “biological sample” includes, but is not limited to, anyquantity of a substance from a living thing or formerly living thing.Such living things include, but are not limited to, humans, mice,monkeys, rats, rabbits, horses, and other animals. Such substancesinclude, but are not limited to, blood, serum, plasma, urine, cells,organs, tissues, bone, bone marrow, lymph, lymph nodes, synovial tissue,chondrocytes, synovial macrophages, endothelial cells, and skin.

Reagents and Immunogens

In constructing an immunoassay, a conjugate of the compound of formulaIV is constructed to compete with the compounds of formula I or formulaII in the sample for binding sites on the antibodies. In the immunoassayof this invention, the reagents are conjugates of a carrier with thecompound of formula IV. In the compound of formula IV the linker spacerconstitutes the “—B—(Y)p-X′—” portion of this molecule. The linker X′and the spacer “—B—(Y)p-”— are conventional in preparing conjugates andimmunogens. Any of the conventional spacer-linking groups utilized toprepare conjugates and immunogens for immunoassays can be utilized inthe compound of formula IV. Such conventional linkers and spacers aredisclosed in U.S. Pat. No. 5,501,987 and U.S. Pat. No. 5,101,015.

Among the preferred spacer groups are included the spacer groupshereinbefore mentioned. Particularly preferred spacing groups are groupssuch as alkylene containing from 1 to 10 carbon atoms,

wherein m and o are integers from 0 to 6, and n is an integer from 1 to6 with alkylene being the especially preferred spacing group In theseformulae m is 0, n is preferably an integer of from 1-6, most preferably1 or 2 and o is preferably 0 or 1.

In the compound of formula IV, X′ is —CH₂— or a functional group linkingthe spacer to the carrier, preferably to an amine group on a polymericcarrier. The group X′ is the result of the terminal functional group Xin the compound of formula III which is capable of binding to a carrier,preferably to an amino group in the polyamine polymer present in thecarrier or used as the immunogen. Any terminal functional group capableof binding to a carrier, preferably capable of reacting with an aminecan be utilized as the functional group X in the compound of formulaIII. These terminal functional groups preferably included within X are:

wherein R₃ is hydrogen or taken together with its attached oxygen atomforms a reactive ester and R₄ is oxygen or sulfur. The radical —N═C═R₄can be an isocyanate or an isothiocyanate. The active esters formed byOR₃ include imidoester, such as N-hydroxysuccinamide, 1-hydroxybenzotriazole and p-nitrophenyl ester. However any active ester whichcan react with an amine group can be used.

The carboxylic group and the active esters are coupled to the carrier orimmunogenic polymer by conventional means. The amine group on thepolyamine polymer, such as a protein, produces an amide group whichconnects the spacer to the polymer, immunogens or carrier and/orconjugates of this invention. On the other hand, carriers can be coatedwith a polyamine polymer to supply the amino group for linking to theligand portion.

In the immunogens and conjugates of the present invention, the chemicalbonds between the carboxyl group-containing the compound of formula IIIas a hapten and the amino groups on the polyamine polymer on the carrieror immunogen can be produced using a variety of methods known to oneskilled in the art. It is frequently preferable to form amide bonds.Amide bonds are formed by first activating the carboxylic acid moiety ofthe hapten in the compounds of formula III by reacting the carboxy groupwith a leaving group reagent (e.g., N-hydroxysuccinimide,1-hydroxybenzotriazole, o- or p-nitrophenol, or o- or p-nitrophenylchloroformate). An activating reagent such as dicyclohexylcarbodiimide,diisopropylcarbodiimide and the like can be used. The activated form ofthe carboxyl group in the hapten of formula III is then reacted with abuffered solution containing the protein carrier. Various methods ofconjugating haptens and carriers are also disclosed in U.S. Pat. No.3,996,344 and U.S. Pat. No. 4,016,146, which are herein incorporated byreference.

Where X is a terminal isocyanate or isothiocyanate radical in thecompound of formula III, these radicals when reacted with the free amineof a polyamine polymer produce the conjugate or the immunogen of thehapten of formula IV where X′ is,

In the ligand of formula IV, X′ functionally connects the hapten withthe amino group on the polyamine containing carrier or on theimmunogenic polypeptide.

Where X, in the compounds of formula III is an aldehyde group thesecompounds may be connected to the amine group of the polyaminepolypeptide or carrier through an amine linkage by reductive amination.Any conventional method of condensing an aldehyde with an amine such asthrough reductive amination can be used to form this linkage. In thiscase, X′ in the ligand portions of formula IV is —CH₂—. Any conventionalmeans of condensing a reactive carbonyl with the amine group can be usedin carrying out this condensation reaction.

The compound of formula III, when B is a methylene carbonyl of theformula:

is produced by condensing the amine group in the compound of formula IIwith an acyl chloride of the formula:Cl—C(═O)—CH₂—(Y)p-X  V

Any conventional method of reacting a primary amine with an acylchloride can be used in this condensation procedure. Where Y is loweralkylene and B is the above methylene carbonyl group in the compound offormula III, this compound is produced by treating the compound offormula II with an anhydride of a di carboxylic acid such as glutaricanhydride. Any conventional means of condensing an anhydride with theprimary amine group can be used in carrying out this condensationreaction

The compound of formula III when B is —CH₂— may be produced by reactingthe compound of formula I with an alkyl halide of the formula:Halo CH₂—(Y)p-X  VII

-   -   wherein Y, p and X are as above.

Any conventional means of condensing an alkyl halide with a primaryamine group can be used in carrying out this condensation reaction.

The compound of formula III where B is —C(═O)—NH—CH₂ may be produced bycondensing the compound of formula II with a halide or the formula:

wherein Y, p and X are as above.utilizing conventional means.

The compound of formula III where B is —C(═O)—O—CH₂— may be produce bycondensing the compound of formula II with a compound of the formula:

where Y, p and X are as above,utilizing conventional means,In cases where the compounds of formula V, VII, VIII, and IX contain areactive amino group as well as a reactive carboxyl group, it isnecessary to use an amine or ester protecting group during the reactionsto form the compounds of formula III. Typically, the amines areprotected by forming the corresponding N-trifluoroacetamide,N-tertbutyloxycarbonyl urethane (N-t-BOC urethane), N-carbobenzyloxyurethane or similar structure. Once the condensation reaction with thestructure of formula I has been accomplished, as described above, theamine or the ester protecting group can be removed using reagents thatdo not otherwise alter the structure of the conjugate. Such reagents andmethods are known to one skilled in the art and include weak or strongaqueous or anhydrous acids, weak or strong aqueous or anhydrous bases,hydride-containing reagents such as sodium borohydride or sodiumcyanoborohydride and catalytic hydrogenation.

The compound of formula III can be converted into the immunogens and/orthe conjugate reagents of this invention by reacting this compound witha carrier, preferably a polyamine polypeptide or a carrier coated with apolyamine polypeptide as described above. The same polypeptide can beutilized as the carrier and as the immunogenic polymer in the immunogenof this invention provided that polyamines or polypeptides areimmunologically active. However, to form the conjugates used as reagentsin the immunoassay, these polymers need not produce an immunologicalresponse as needed for the immunogens. In accordance with thisinvention, the various functional group represented by X in thecompounds of formula III can be conjugated to the carrier byconventional means of attaching a functional group to a carrier. Inaccordance with a preferred embodiment, in the compounds of formula III,X is a carboxylic acid group or an activated carboxyl group.

Antibodies

The present invention also relates to novel antibodies, particularlymonoclonal antibodies, to the compounds of formula I and formula II andmixtures thereof which can be produced by utilizing the aforementionedimmunogens. It has been found that these antibodies produced inaccordance with this invention are selectively reactive with thecompounds of formula I and the compound of formula II and mixturesthereof. These antibodies do not react with non-pharmaceuticallyinactive metabolites of the compounds of formula I and the compound offormula II which would interfere with immunoassays for either thecompound of formula I and the compound of formula II. The ability of theantibodies of this invention not to react with these inactivemetabolites makes these antibodies particularly valuable in providing animmunoassay for either the compound of formula I or the compound offormula II.

The present invention relates to these selectively reactive novelantibodies to the compounds of formula I and formula II and mixturesthereof. The antisera of the invention can be conveniently produced byimmunizing host animals with the immunogens of this invention. Suitablehost animals include rodents, such as, for example, mice, rats, rabbits,guinea pigs and the like, or higher mammals such as goats, sheep, horsesand the like. Initial doses, bleedings and booster shots can be givenaccording to accepted protocols for eliciting immune responses inanimals. Through periodic bleeding, the blood samples of the immunizedmice were observed to develop an immune response against the compoundsof formula I and II utilizing conventional immunoassays. These methodsprovide a convenient way to screen for hosts and antibodies which areproducing antisera having the desired activity.

The antibodies having substantially 100% selective reactivity withlenalidomide and substantially no selective reactivity with thalidomideor substantially selective reactivity with both thalidomide andlenalidomide can be produced utilizing the immunogen of formula III andby the screening method disclosed below. This screening method can beused to obtain antibodies which are reactive with both the lenalidomideand thalidomide chemotherapeutic agents, antibodies which are specificand selective to lenalidomide and antibodies having any desired relativereactivity with regard to these chemotherapeutic agents.

In preparing these antibodies, an immunogenic carrier can be conjugatedwith the immunogen of formula III and used to immunize host animals suchas mice, rabbits, sheep or rats. Development of the immune response tothe compound of formula III can be monitored by ELISA utilizingmicrotiter plates coated with a conjugate of BSA and the compound offormula III. Once the immune response has been sufficiently developedthe spleen cells of the host animal can be isolated and fused with animmortalized cell line. With respect to producing monoclonal antibodiesthe fused cells can be plated on 96-well plates and grown in thepresence of a selective medium to select hybridoma cells. Hybridomasupernatants and antisera can be assayed for the presence ofanti-lenalidomide antibodies by ELISA. Antibodies from wells that gavepositive ELISA results can be tested for lenalidomide and thalidomidebinding by indirect competitive microtiter plate assay. The IC₅₀ valuesof an analyte such as lenalidomide and thalidomide and theirmetabolites, can be calculated from this assay. The IC₅₀ (inhibitoryconcentration at 50%) of an analyte in an assay is the concentration ofthat analyte in a sample at which the signal in the assay is 50% of thetotal signal for the assay in the absence of analyte in an inhibitionassay.—Selective reactivity of an analyte is calculated from a ratio ofthe IC₅₀'s expressed as a %: 100%−[IC₅₀-analyte/(IC^(l)₅₀-lenalidomide+IC^(l) ₅₀-thalidomide)]×100. For antibodies havingsubstantially 100% selective reactivity with lenalidomide andsubstantially no selective reactivity with thalidomide this value willapproach 100%. The calculation of the IC₅₀ is carried out according tothe procedure found in The Immunoassay Handbook, pp 108-110, 3^(rd)edition, edited by D. Wild, published by Elsevier, Amsterdam, 2005. Asseen from the above formula, the IC₅₀ of an analyte is inverselyproportional to the reactivity of the analyte. Cells from wells that haddesired relative reactivity with both lenalidomide and thalidomide canbe obtained by screening and sub-cloning by limiting dilution to isolateindividual clones producing monoclonal antibodies having the desiredreactivity with thalidomide and lenalidomide

Monoclonal antibodies are produced conveniently by immunizing Balb/cmice according to the schedule followed by injecting the mice withadditional immunogen i.p. or i.v. on three successive days startingthree days prior to the cell fusion. Other protocols well known in theantibody art may of course be utilized as well. The completeimmunization protocol detailed herein provided an optimum protocol forserum antibody response for the antibody to the compounds of formula Iand II.

B lymphocytes obtained from the spleen, peripheral blood, lymph nodes orother tissue of the host may be used as the monoclonal antibodyproducing cell. Most preferred are B lymphocytes obtained from thespleen. Hybridomas capable of generating the desired monoclonalantibodies of the invention are obtained by fusing such B lymphocyteswith an immortal cell line, which is a cell line that imparts long termtissue culture stability on the hybrid cell. In the preferred embodimentof the invention the immortal cell may be a lymphoblastoid cell or aplasmacytoma cell such as a myeloma cell. Murine hybridomas whichproduce lenalidomide and thalidomide monoclonal antibodies are formed bythe fusion of mouse myeloma cells and spleen cells from mice immunizedwith the aforementioned immunogenic conjugates. Chimeric and humanizedmonoclonal antibodies can be produced by cloning the antibody expressinggenes from the hybridoma cells and employing recombinant DNA methods nowwell known in the art to either join the subsequence of the mousevariable region to human constant regions or to combine human frameworkregions with complementary determining regions (CDR's) from a donormouse or rat immunoglobulin. An improved method for carrying outhumanization of murine monoclonal antibodies which provides antibodiesof enhanced affinities is set forth in International Patent ApplicationWO92/11018.

Polypeptide fragments comprising only a portion of the primary antibodystructure may be produced, which fragments possess one or moreimmunoglobulin activities. These polypeptide fragments may be producedby proteolytic cleavage of intact antibodies by methods well known inthe art, or by inserting stop codons at the desired locations inexpression vectors containing the antibody genes using site-directedmutageneses to produce Fab fragments or (Fab′)₂ fragments. Single chainantibodies may be produced by joining VL and VH regions with a DNAlinker (see Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85:5879-5883(1988) and Bird et al., Science, 242:423-426 (1988))

The antibodies produced in accordance with this invention can beselectively reactive with the compound of formula II or both thecompounds of formula I and the compound of formula II without having anysubstantial cross-reactivity with the pharmacologically, therapeuticallynon-active metabolites of the compound of formula I and the compound offormula II. By having no substantial cross-reactivity, it is meant thatthe antibodies of this invention have cross-reactivity relative to boththeir reactivity with the compound of formula I and the compound offormula II of less than 10%, preferably less than 5%.

In accordance with this invention antibodies which are selectivelyreactive with lenalidomide and have no selective reactivity withthalidomide can be produced. By an antibody having selective reactivityfor lenalidomide and with no selective reactivity with thalidomide, itis meant that the antibody has at least 95% activity for lenalidomidebased upon its reactivity or binding with both thalidomide andlenalidomide. In order to be utilized in a thalidomide immunoassay theantibody should be selectively reactive with both lenalidomide andthalidomide and have a cross-reactivity with the aforementionedpharmacologically, therapeutically non-active metabolites of thecompound of formula I and the compound of formula II of less than 10%based upon its reactivity with the both the compounds of formula I andformula II and have a reactivity with thalidomide of at least 10% basedupon its reactivity with both thalidomide and lenalidomide. In order tobe utilized in a lenalidomide immunoassay, any antibody of thisinvention which is selectively reactive with lenalidomide or isselectively reactive with both lenalidomide and thalidomide and have across-reactivity with the aforementioned pharmacologically,therapeutically non-active metabolites of the compound of formula I andthe compound of formula II of less than 10% based upon its reactivitywith the both the compounds of formula I and formula II can be used.

Immunoassays

In accordance with this invention, the conjugates and the antibodiesgenerated from the immunogens of the compound of formula III can beutilized as reagents for the determination of the compounds of formula Iand formula II in patient samples. This determination is performed bymeans of an immunoassay. Any immunoassay in which the reagent conjugatesformed from the compound of formula III compete with the compound offormula I or formula II in the sample for binding sites on theantibodies generated in accordance with this invention can be utilizedto determine the presence of the compound of formula I or formula II ina patient sample. The manner for conducting such an assay for thecompound of formula I and formula II in a sample suspected of containinglenalidomide or thalidomide, comprises combining an (a) aqueous mediumsample, (b) an antibody to the compound of formula I and formula IIgenerated in accordance with this invention and (c) the conjugatesformed from the compound of formula III. The compound of formula I orformula II in the sample can be determined by measuring the inhibitionof the binding to the specific antibody of a known amount of theconjugate added to the mixture of the sample and antibody. The result ofthe inhibition of such binding of the known amount of conjugates by theunknown sample is compared to the results obtained in the same assay byutilizing known standard solutions of the compounds of formula I orformula II. In determining the amount of the compounds of formula I orformula II in an unknown sample, the sample, the conjugates formed fromthe compounds of formula III and the antibody may be added in any order.

Various means can be utilized to measure the amount of conjugate formedfrom the compound of formula III bound to the antibody. One method iswhere binding of the conjugates to the antibody causes a decrease in therate of rotation of a fluorophore conjugate. The amount of decrease inthe rate of rotation of a fluorophore conjugate in the liquid mixturecan be detected by the fluorescent polarization technique such asdisclosed in U.S. Pat. No. 4,269,511 and U.S. Pat. No. 4,420,568.

On the other hand, the antibody can be coated or absorbed onnanoparticles so that when these particles react with the compounds offormula I or formula II and conjugates formed from the compounds offormula III these nanoparticles form an aggregate. However, when theantibody coated or absorbed on nanoparticles react with thalidomide orlenalidomide in the sample, the thalidomide or lenalidomide from thesample bound to these nanoparticles does not cause aggregation of theantibody nanoparticles. The amount of aggregation or agglutination canbe measured in the assay mixture by absorbance.

On the other hand, these assays can be carried out by having either theantibody or the compounds of formula III attached to a solid supportsuch as a microtiter plate or any other conventional solid supportincluding solid particles. Attaching antibodies and proteins to suchsolid particles is well known in the art. Any conventional method can beutilized for carrying out such attachments. In many cases, in order toaid measurement, labels may be placed upon the antibodies, conjugates orsolid particles, such as radioactive labels or enzyme labels, as aids indetecting the amount of the conjugates formed from the compound offormula III which is bound or unbound with the antibody. Other suitablelabels include chromophores, fluorophores, etc.

As a matter of convenience, assay components of the present inventioncan be provided in a kit, a packaged combination with predeterminedamounts of new reagents employed in assaying for the compounds offormula I or formula II. These reagents include the antibody of thisinvention, as well as, the conjugate formed from the compounds offormula III. In carrying out an immunoassay in accordance with thisinvention the radicals p, X, Y and B in the reagent and the immunogenwhich forms the antibody used in a given immunoassay can be the same orbe a different substituent within the groups defined for each of theseradicals. Therefore while the definitions of the radicals p, X, Y, and Bare the same for the conjugate reagent and the immunogen, the particularsubstituent which these radicals represent for the immunogen and theconjugate reagent in a given assay may be different.

In addition to these necessary reagents, additives such as ancillaryreagents may be included, for example, stabilizers, buffers and thelike. The relative amounts of the various reagents may vary widely toprovide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. Reagents can beprovided in solution or as a dry powder, usually lyophilized, includingexcipients which on dissolution will provide for a reagent solutionhaving the appropriate concentrations for performing the assay.

EXAMPLES

In the examples, the following abbreviations are used for designatingthe following:

-   -   HATU O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium        hexafluorophosphate    -   DIPEA N—N′-Diisopropylethylamine    -   DMF Dimethylformamide    -   TFA Trifluoroacteic acid    -   CH₂Cl₂ dicholoromethane    -   DMSO Dimethylsulfoxide    -   NHS N-hydroxy succinimide    -   s-NHS sulfo-N-hydroxy succinimide    -   EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride    -   KLH Keyhole Limpet Hemocyanin    -   BSA Bovine serum albumin    -   PBS Phosphate buffered saline    -   NaCl sodium chloride    -   HRP horseradish peroxidase    -   ANS 8-Anilino-1-naphthalenesulfonic acid    -   TMB 3,3′,5,5′-Tetramethylbenzidine    -   TRIS Tris(hydroxymethyl)aminomethane hydrochloride    -   di H₂O deionized water

The phosphate buffer composition has an aqueous solution containing

-   -   15.4 mM Sodium phosphate dibasic (Na₂HPO₄)    -   4.6 mM Sodium phosphate monobasic (NaH₂PO₄)    -   pH=7.2±0.10

In the examples, Schemes 1-2 below set forth the specific compoundsprepared and referred to by numbers in the Examples. The schemes are asfollows:

Example 1 Preparation of Carbamoyl Pentantoic Acid Derivative ofLenalidomide Derivative [3] (Scheme 1)

A mixture of lenalidomide [1] (1.0 g, 3.86 mmol) and glutaric anhydride[2] (0.48 g, 4.25 mmol) in anhydrous toluene was heated and refluxedunder nitrogen for 3.5 hours. Another portion of glutaric anhydride [2](0.18 g, 1.53 mmol) was added and the mixture was heated another 2 hoursto produce [3]. The mixture was cooled to 0° C., to precipitate [3]. Theprecipitated solid was filtered, and washed with CH₂Cl₂ to obtain 1.55 gof crude compound [3]. This crude compound was recrystallized fromethanol (20 mL)/H₂O (1 mL) to obtain pure [3] (1.30 g, 90%) as a whitesolid.

Example 2 Preparation of Carbamoyl-Butyrylamino-Methyl Benzoic AcidDerivative of Lenalidomide Derivative [7] (Scheme 2)

The compound [3] produced in example 1 (800 mg, 2.14 mmol) was dissolvedin anhydrous DMF (20 mL) under nitrogen, to which was addeddiisopropylethyl amine (DIPEA) (1.27 mL, 7.27 mmol) and the amine [10](700 mg, 2.35 mmol) followed by HATU (1.88 g, 4.93 mmol). This reactionmixture was stirred at 25° C. for 24 hours to produce [6]. The contentsof the flask were diluted with ethyl acetate. The organic phase (ethylacetate) was washed with 1 M hydrochloric acid, saturated sodiumbicarbonate and water. The ethylacetate layer was then dried over sodiumsulfate, which was filtered off. Removal of the ethylacetate solventprovided the crude product [6], which was purified by flashchromatography with 100% EtOAc and 1-2% MeOH/EtOAc to obtain the pureproduct [6] (680 mg, 57%) as a white solid.

¹H NMR (300 MHz, DMSO-d₆): δ 11.03 (s, 1H), 9.82 (s, 1H), 8.46 (t, J=6.0Hz, 1H), 7.82-7.86 (m, 3H), 7.46-7.52 (m, 2H), 7.35 (d, J=8.3 Hz, 2H),5.14 (dd, J=5.0, 13.5 Hz, 1H), 4.32-4.38 (m, 4H), 2.86-2.96 (m, 1H),2.56-2.64 (m, 2H), 2.39 (t, J=7.5 Hz, 2H), 2.25 (t, J=7.5 Hz, 2H),1.97-2.05 (m, 1H), 1.82-1.92 (m, 2H), 1.53 (s, 9H). APCI]⁻=561.

This white solid [6] (676 mg, 1.20 mmol) was dissolved indichloromethane (3 mL). To this solution of compound [6] at 0° C. underN₂ was added TFA (3 mL) to produce [7]. The dichloromethane was removedunder reduced pressure and the resulting residue [7] was triturated withether to isolate the crude acid. This material was recrystallized fromaqueous EtOH to obtain pure [7] (507 mg, 83%).

Example 3 General Method for Preparing NHS/s-NHS Activated DrugDerivatives from the Corresponding Acids [3] & [7]

Lenalidomide acid derivative [3] was activated with EDC and NHS toproduce the NHS activated ester of lenalidomide [4] for eventualconjugation to proteins (examples 4 and 5a). Lenalidomide acidderivative [7] was activated with EDC and s-NHS to produce the s-NHSactivated ester of lenalidomide [8] for eventual conjugation to protein(example 5b).

Example 3a Preparation of NHS Activated Ester Lenalidomide CarbamoylPentantoic Acid Derivative [4]

Lenalidomide derivative [3], example 1, scheme 1, (67.62 mg) wasdissolved in 7 mL of DMSO to which was added NHS (59.60 mg) and EDC(93.00 mg). The reaction mixture was stirred for 20 hours at ambienttemperature under a nitrogen atmosphere to produce the NHS activatedester of lenalidomide derivative [4]. The reaction mixture was useddirectly in examples 4 and 5a.

Example 3b Preparation of s-NHS Activated Ester LenalidomideCarbamoyl-Butyrylamino-Methyl Benzoic Acid Derivative [8]

Lenalidomide derivative [7], example 2, scheme 2 (16.3 mg) was dissolvedin 1.6 mL of DMSO to which was added s-NHS (25.3 mg) and EDC (18.1 mg).The reaction mixture was stirred for 20 hours at ambient temperatureunder a nitrogen atmosphere to produce the s-NHS activated ester oflenalidomide derivative [8]. The reaction mixture was used directly inexample 5b.

Example 4 Preparation of KLH Immunogen with Activated Hapten [4]

A protein solution of KLH was prepared by dissolving 300 mg of KLH in 15mL of phosphate buffer (50 mM, pH 7.5), followed by addition of 1.5 mLDMSO and 3.50 mL of NHS activated lenalidomide derivative [4] preparedin Example 3a. The reaction mixture of KLH and activated lenalidomidederivative [4] was allowed to stir for 20 hours at room temperature toproduce the lenalidomide-KLH conjugate [5]. The lenalidomide-KLHconjugate [5] was then purified by dialysis against 10% DMSO inphosphate buffer (50 mM, pH 7.5) at room temperature. Thereafterlenalidomide-KLH conjugate [5] was dialyzed against phosphate buffer (50mM, pH 7.5) at room temperature. The last dialysis was performed againstphosphate buffer at 4° C. The lenalidomide-KLH conjugate [5] wascharacterized by ultraviolet-visible spectroscopy (UV/VIS). Theconjugate was diluted to a final concentration of 2 mg/mL in phosphatebuffer (50 mM, pH 7.5).

Example 5a Preparation of BSA Conjugate with Activated Hapten [4]

A protein solution of BSA was prepared by dissolving 1 g BSA inphosphate buffer (50 mM, pH 7.5) for a final concentration of 50 mg/mL.DMSO (3.3 mL) was slowly added to the protein solution of BSA whilestirring on ice, followed by addition of 0.60 mL of NHS activatedlenalidomide derivative [4] prepared in Example 3a. The amount of NHSactivated lenalidomide derivative [4] added to the protein solution ofBSA was calculated for a 1:1 molar ratio between the derivative oflenalidomide [4] and BSA. The mixture of BSA and activated lenalidomidederivative [4] was allowed to stir for 18 hours at room temperature toproduce the conjugate of the activated lenalidomide ester [4] and BSA.This conjugate was then purified by dialysis against 10% DMSO inphosphate buffer (50 mM, pH 7.5) at room temperature. Thereafterlenalidomide-BSA conjugate [5] was dialyzed against phosphate buffer (50mM, pH 7.5) at room temperature. The last dialysis was performed againstphosphate buffer at 4° C. The purified lenalidomide-BSA conjugate [5]was characterized by UV/VIS spectroscopy.

Example 5b Preparation of BSA Conjugate with Activated Hapten [8]

A protein solution of BSA was prepared by dissolving 0.5 g BSA inphosphate buffer (50 mM, pH 7.5) for a final concentration of 50 mg/mL.s-NHS activated lenalidomide derivative [8] prepared in Example 3b wasslowly added to the protein solution of BSA while stirring on ice. Theamount of s-NHS activated lenalidomide derivative [8] added to theprotein solution of BSA was calculated for a 1:1 molar ratio between thederivative of lenalidomide [8] and BSA. The mixture of BSA and activatedlenalidomide derivative [8] was allowed to stir for 18 hours at roomtemperature to produce the conjugate of the activated lenalidomide ester[8] and BSA. This conjugate was then purified by dialysis against 10%DMSO in phosphate buffer (50 mM, pH 7.5) at room temperature. Thereafterlenalidomide-BSA conjugate [9] was dialyzed against phosphate buffer (50mM, pH 7.5) at room temperature. The last dialysis was performed againstphosphate buffer at 4° C. The purified lenalidomide-BSA conjugate [9]was characterized by UV/VIS spectroscopy.

Example 6a Preparation of Polyclonal Antibodies to Lenalidomide [3]

Ten female BALB/c mice were immunized i.p. with 100 μg/mouse oflenalidomide-KLH immunogen [5], as prepared in Example 4, emulsified inComplete Freund's adjuvant. The mice were boosted once, four weeks afterthe initial injection with 100 μg/mouse of the same immunogen emulsifiedin Incomplete Freund's Adjuvant. Twenty days after the boost, testbleeds containing polyclonal antibodies from each mouse were obtained byorbital bleed. The anti-serum from these test bleeds containinglenalidomide antibodies were evaluated in Examples 8 and 9.

Example 6b Preparation of Monoclonal Antibodies to Lenalidomide [3]

Mice from Example 6a that were immunized with lenalidomide-KLH conjugate[5] prepared in Example 4 were used to produce monoclonal antibodies.For monoclonal antibodies starting three days before the fusion, themice were injected i.p. with 400 μg (3 days before fusion), 200 μg (2days before fusion), and 200 μg (1 day before fusion) oflenalidomide-KLH conjugate [5] in PBS prepared in Example 4. Spleencells were isolated from the selected mice and fused with 2×10⁷ SP2/0cells with 50% polyethylene glycol 1500 according to the method ofColigan, J. E. et al., eds., Current Protocols in Immunology,2.5.1-2.5.8, (1992), Wiley & Sons, NY. The fused cells were plated onten 96-well plates in DMEM/F12 supplemented with 20% FetalClone I, 2%L-glutamine (100 mM) and 2% 50×HAT. Two to three weeks later, thehybridoma supernatant was assayed for the presence of anti-lenalidomideantibodies by ELISA (as in example 8b). Cells from the wells that gavepositive ELISA results were expanded to 24 well plates. These monoclonalantibodies were tested for lenalidomide and thalidomide binding byindirect competitive microtiter plate assay as described in example 9.Clones positive by ELISA were subcloned at least once by limitingdilution according to the method disclosed in Coligan, J. E. et al.,eds., Current Protocols in Immunology, 2.5.8-2.5.17, (1992), Wiley &Sons, NY.

Example 7a Microtiter Plate Sensitization Procedure withLenalidomide-BSA Conjugate [5]

The ELISA method for measuring lenalidomide concentrations was performedin polystyrene microtiter plates (Nunc MaxiSorp F8 Immunomodules)optimized for protein binding and containing 96 wells per plate. Eachwell was coated with lenalidomide-BSA conjugate [5] (prepared as inExample 5a) by adding 300 μL of lenalidomide-BSA conjugate [5] at 10μg/mL in 0.05M sodium carbonate, pH 9.6, and incubating for three hoursat room temperature. The wells were washed with 0.05M sodium carbonate,pH 9.6 and then were blocked with 375 μL of 5% sucrose, 0.2% sodiumcaseinate solution for 30 minutes at room temperature. After removal ofthe post-coat solution the plates were dried at 37° C. overnight.

Example 7b Microtiter Plate Sensitization Procedure withLenalidomide-BSA Conjugate [9]

The ELISA method for measuring lenalidomide concentrations was performedin polystyrene microtiter plates (Nunc MaxiSorp F8 Immunomodules)optimized for protein binding and containing 96 wells per plate. Eachwell was coated with lenalidomide-BSA conjugate [9] (prepared as inExample 5b) by adding 300 μL of lenalidomide-BSA conjugate [9] at 10μg/mL in 0.05M sodium carbonate, pH 9.6, and incubating for three hoursat room temperature. The wells were washed with 0.05M sodium carbonate,pH 9.6 and then were blocked with 375 μL of 5% sucrose, 0.2% sodiumcaseinate solution for 30 minutes at room temperature. After removal ofthe post-coat solution the plates were dried at 37° C. overnight.

Example 8a Antibody Screening Procedure—Titer

This procedure is to find the dilution of antibody to be tested fordisplacement as in Example 9. The ELISA method for screeninglenalidomide antibodies (produced in Example 6) was performed with themicrotiter plates that were sensitized with lenalidomide-BSA conjugatesprepared in Examples 7a and 7b. The antibody screening assay wasperformed by diluting the murine serum from test bleeds (as in Example6a) containing polyclonal lenalidomide antibodies to 1:2,000, 1:6,000,1:18,000 and 1:54,000 (volume/volume) in phosphate buffered salinecontaining 0.1% BSA and 0.01% thimerosal. To each well oflenalidomide-BSA sensitized wells (prepared in Examples 7a and 7b) 50 μLphosphate buffered saline containing 0.1% BSA and 0.01% thimerosal and50 μL of diluted antibody were added and incubated for 10 minutes atroom temperature with shaking. During this incubation antibody binds tothe lenalidomide-BSA conjugate passively absorbed in the wells (Examples7a and 7b). The wells of the plates were washed three times with 0.02 MTRIS, 0.9% NaCl, 0.5% Tween-80 and 0.001% thimerosal, pH 7.8 to removeany unbound antibody. To detect the amount of lenalidomide antibodybound to the lenalidomide-BSA conjugate in the wells, 100 μL of a goatanti-mouse antibody—HRP enzyme conjugate (Jackson Immunoresearch)diluted to a specific activity (approximately 1/3000) in PBS with 0.1%BSA, 0.05% ANS, 0.01% thimerosal, capable of binding specifically withmurine immunoglobulins and producing a colored product when incubatedwith a substrate, in this example TMB, were added to each well. After anincubation of 10 minutes at room temperature with shaking, during whichthe goat anti-mouse antibody—HRP enzyme conjugate binds to lenalidomideantibodies in the wells, the plates were again washed three times toremove unbound goat anti-mouse antibody—HRP enzyme conjugate. To developa measurable color in the wells washing was followed by the addition of100 μL of TMB (TMB Substrate, BioFx), the substrate for HRP, to developcolor during a 10 minute incubation with shaking at room temperature.Following the incubation for color development, 50 μL of stop solution(1.5% sodium fluoride in di H₂O) was added to each well to stop thecolor development and after 20 seconds of shaking the absorbance wasdetermined at 650 nm (Molecular Devices Plate Reader). The amount ofantibody in a well was proportional to the absorbance measured and wasexpressed as the dilution (titer) resulting in an absorbance of 1.5.Titers were determined by graphing antibody dilution of the antibodymeasured (x-axis) vs. absorbance 650 nm (y-axis) and interpolating thetiter at an absorbance of 1.5. The titer which produced absorbance of1.5 determined the concentration (dilution) of antibody used in theindirect competitive microtiter plate assay described in Example 9.

Example 8b Antibody Screening Procedure—Monoclonal Screening

The ELISA method for screening lenalidomide monoclonal antibodies(produced in example 8b) was performed with the microtiter plates thatwere sensitized with lenalidomide-BSA conjugate [9] as described inexample 7b. To each well of lenalidomide-BSA sensitized wells (preparedin example 7b) 50 μL phosphate buffered saline containing 0.1% BSA and0.01% thimerosal and then 50 μL of monoclonal culture supernatant wereadded and incubated for 10 minutes at room temperature with shaking.During this incubation antibody binds to the lenalidomide-BSA conjugatein the well. The wells of the plates were washed three times with 0.02 MTRIS, 0.9% NaCl, 0.5% Tween-80 and 0.001% thimerosal, pH 7.8 to removeany unbound antibody. To detect the amount of lenalidomide antibodybound to the lenalidomide-BSA conjugate in the wells, 100 μL of a goatanti-mouse antibody—HRP enzyme conjugate (Jackson Immunoresearch)diluted 1/3000 in PBS with 0.1% BSA, 0.05% ANS, 0.01% thimerosal,capable of binding specifically with murine immunoglobulins andproducing a colored product when incubated with a substrate, in thisexample TMB, were added to each well. After an incubation of 10 minutesat room temperature with shaking, during which the goat anti-mouseantibody—HRP enzyme conjugate binds to lenalidomide antibodies in thewells, the plates were again washed three times to remove unbound goatanti-mouse antibody—HRP enzyme conjugate. To develop a measurable colorin the wells washing was followed by the addition of 100 μL of TMB (TMBSubstrate, BioFx), the substrate for HRP, to develop color during a 10minute incubation with shaking at room temperature. Following theincubation for color development, 50 μL of stop solution (1.5% sodiumfluoride in diH₂O) was added to each well to stop the color developmentand after 10 seconds of shaking the absorbance was determined at 650 nm(Molecular Devices Plate Reader). The amount of antibody in a well wasproportional to the absorbance measured. Samples with an absorbance ofgreater than three or more times background were designated as positive.Samples with absorbance above 0.4 or fifty samples with highestabsorbance were expanded to 24 well plates, as described in Example 8b.

Example 9 Indirect Competitive Microtiter Plate Immunoassay ProcedureDetermining IC₅₀ for Antibodies to Lenalidomide

The ELISA method for determining IC₅₀ values was performed with themicrotiter plates that were sensitized with lenalidomide-BSA conjugate[9] as described in Example 7b. The analytes—lenalidomide andthalidomide were dissolved in DMSO and diluted in diH₂O over aconcentration range of 1 to 100,000 ng/mL. Each of the assays wereperformed by incubating 50 μL of the analyte solution with 50 μL of oneof the antibodies selected from the polyclonal antibodies produced inExample 6a with the immunogen of Example 4 (lenalidomide) and themonoclonal antibody produced in Example 8b (lenalidomide andthalidomide). The assays were all performed by diluting theconcentration of the antibodies in each of the wells to the titerdetermined in Example 8a. During the 10 minute incubation (at roomtemperature with shaking) there is a competition of antibody binding forthe lenalidomide-BSA conjugate in the well (produced in Example 7b) andthe analyte in solution. Following this incubation the wells of theplate were washed three times with 0.02 M TRIS, 0.9% NaCl, 0.5% Tween-80and 0.001% thimerosal, pH 7.8 to remove any material that was not bound.To detect the amount of lenalidomide antibody bound to thelenalidomide-BSA conjugate in the wells (produced in Example 7b), 100 μLof a goat anti-mouse antibody—HRP enzyme conjugate (JacksonImmunoresearch) diluted to a predetermined specific activity(approximately 1/3000) in PBS with 0.1% BSA, 0.05% ANS, 0.01%thimerosal, capable of binding specifically with murine immunoglobulinsand producing a colored product when incubated with a substrate, in thisexample TMB, were added to each well. After an incubation of 10 minutesat room temperature with shaking, during which the goat anti-mouseantibody—HRP enzyme conjugate binds to lenalidomide antibodies in thewells, the plates were again washed three times to remove unboundsecondary conjugate. To develop a measurable color in the wells washingwas followed by the addition of 100 μL of TMB (TMB Substrate, BioFx),the substrate for HRP, to develop color in a 10 minute incubation withshaking at room temperature. Following the incubation for colordevelopment, 50 μL of stop solution (1.5% sodium fluoride in di H₂O) wasadded to each well to stop the color development and after 20 seconds ofshaking the absorbance was determined at 650 nm (Molecular Devices PlateReader). The amount of antibody in a well was proportional to theabsorbance measured and inversely proportional to the amount oflenalidomide or thalidomide in the sample. The IC₅₀'s of lenalidomideand thalidomide were determined by constructing dose-response curveswith the absorbance in the wells plotted versus analyte concentration inthe wells. The absorbance of the color in the wells containing analytewas compared to that with no analyte and a standard curve was generated.The IC₅₀ value for a given analyte was defined as the concentration ofanalyte that was required to have 50% of the absorbance of the wellscontaining no analyte. Results for polyclonal antibodies to lenalidomideare in table I below. Results for monoclonal antibodies to lenalidomideare in table II below.

TABLE I IC₅₀'s of lenalidomide and titers of polyclonal antibodies tolenalidomide (Example 6a) using plates coated with lenalidomide-BSAconjugate [9] (Example 7b). Bleed # Titer IC₅₀, ng/mL 1 51,000 84 266,000 59 3 28,000 3,400 4 15,000 2,200 5 111,000 510 6 5,200 810 79,300 80 8 9,400 870 9 58,000 570 10 18,000 9

TABLE II IC₅₀'s of lenalidomide and thalidomide using monoclonalantibodies to lenalidomide (Example 6b) using plates coated withlenalidomide-BSA conjugate [9] (Example 7b). Monoclonal antibody Analyte% cross-reactivity to number Lenalidomide Thalidomide Thalidomide 1H12 17 <15% 6G1 11 992 <2% 7E4 6 7 <86%

As seen from these tables, the antibodies of this invention aresubstantially reactive with lenalidomide and thalidomide.

What is claimed:
 1. An immunoassay for detecting lenalidomide in asample comprising providing a mixture containing said sample, aconjugate of a carrier with a ligand having the formula:

wherein B is —C(═O)—CH₂—, —C(═O)—NH—CH₂—, —C(═O)—O—CH₂— or —CH₂—; Y isan organic spacing group; p is an integer from 0 to 1; and X is aterminal functional group capable of binding to a carrier; and anantibody which is selectively reactive with the pharmaceutically activechemotherapeutic drugs selected from the group consisting oflenalidomide and mixtures of lenalidomide with thalidomide and has nosubstantial cross-reactivity with the non-pharmaceutically activemetabolites of said chemotherapeutic drugs, said cross reactivity beingless than 10% relative to the antibody's reactivity with saidchemotherapeutic drugs and causing the lenalidomide in said sample andsaid conjugate to bind with said antibody and thereafter measuring theamount of said conjugate in said mixture that is bound or unbound tosaid antibody, whereby the presence of lenalidomide in said sample canbe detected.
 2. The immunoassay of claim 1 wherein the sample is a humansample.
 3. The immunoassay of claim 2 wherein said human sample is asample taken from a patient treated with lenalidomide and saidimmunoassay measures the amount of lenalidomide in the sample bymeasuring the amount of conjugate which is bound or unbound to saidantibody.
 4. The immunoassay of claim 3 wherein said antibody has aselective reactivity with lenalidomide of about 100% based upon itsreactivity with said chemotherapeutic drugs.
 5. The immunoassay of claim1 wherein said antibody is generated from an immunogen comprising animmunogenic carrier containing polyamine polymer conjugated with aligand of the formula:

wherein Y and B are as above; and X is a terminal functional groupcapable of binding to said polyamine polymer.
 6. The immunoassay ofclaim 5, wherein the antibody is attached to a solid support.
 7. Theimmunoassay of claim 6, wherein the solid support is microtitor plates.8. The immunoassay of claim 6, wherein the solid support isnanoparticles.
 9. The immunoassay of claim 2, wherein said antibody is amonoclonal antibody.
 10. The immunoassay of claim 9, wherein saidantibody is derived from mice, rabbits, sheep or rats.
 11. A kit fordetermining the presence of lenalidomide in a patient sample comprisingseparate reagents packaged in separate containers, one of said reagentsbeing a conjugate of a carrier with a ligand of the formula

wherein B is —C(═O)—CH₂—, —C(═O)—NH—CH₂—, —C(═O)—O—CH₂— or —CH₂— Y is anorganic spacing group; and p is an integer from 0 to 1; X is a terminalfunctional group capable of binding to a carrier; and the other reagentbeing an antibody selectively reactive with a pharmaceutically activechemotherapeutic drug selected from the group consisting oflenalidomide, and mixtures of thalidomide and lenalidomide and has nosubstantial cross-reactivity with the non-pharmaceutically activemetabolites of said chemotherapeutic drugs, said cross reactivity beingless than 10% relative to the antibody's reactivity with saidchemotherapeutic drugs.
 12. The kit of claim 11 wherein p is
 1. 13. Thekit of claim 12 where Y is lower alkylene.
 14. The kit of claim 13 whereX is

wherein R₃ is hydrogen or taken together with its attached oxygen atomforms a reactive ester and R₄ is oxygen or sulfur.
 15. The kit of claim14, wherein X is


16. The kit of claim 12 wherein Y is

wherein n and o are integers from 0 to 6, and m is an integer from 1 to6.
 17. The kit of claim 16, wherein Y is


18. The kit of claim 17 where X is

wherein R₃ is hydrogen or taken together with its attached oxygen atomforms a reactive ester and R₄ is oxygen or sulfur.
 19. The kit of claim18, wherein X is